Molecular and micellar associations in the pH-dependent stable and metastable dissolution of unconjugated bilirubin by bile salts

Unconjugated bilirubin (UCB) is almost insoluble in water at neutral pH, but appears in normal human gallbladder bile at concentrations up to 35 microM. We therefore determined whether conjugated bile salts could increase the dissolved concentration [( Bt]) of UCB over the pH range 3.0-11.0. Using crystalline UCB, [Bt] was higher with less ordered crystals, with increasing pH and bile salt concentration, and with taurocholate (TC) micelles compared to taurodehydrocholate (TDHC) dimers. Plots of [Bt] verus pH from pH 3.0-9.3 fit the equation, [Bt] = A(1 + K'1/[H]+ + K'1.K'2/[H+]2), where A = [Bt] at pH less than 4.0, and K'1 and K'2 are the two apparent ionization constants of UCB. Estimated pK'1 values in NaCl, TC, and TDHC were 6.8, 6.0, and 5.6, respectively; pK'2 was greater than or equal to 9.3 in each system. Acidification of disodium bilirubinate to pH less than 8.5 produced high, metastable [Bt] in 50 mM TC; this was absent in 0.15 M NaCl, and minor in 50 mM TDHC. In all solutions, maximum [Bt] of 60-65 mM was attained at pH greater than or equal to 10.5. This work helps explain the immense variation among reported [Bt] values, indicates that UCB monoanion predominates at the pH range of bile, and suggests that bile salt monomers, dimers, and micelles enhance the solubility of UCB in bile.     

Influence of bile salts on the endogenous excretion of bile pigments

Effect of the infusion of glycodeoxycholate (GDC), taurocholate (TC) and dehydrocholate (DHC) on bile flow and on bile salt, biliary lipid and bile pigment secretion, has been studied in pentobarbital-anesthetized rabbits. GDC increased bile flow the most, while DHC increased it more than TC. The different choleretic actions of these bile salts cannot be explained by means of variations in their capacity to form micelles. Only GDC and TC were able to stimulate biliary lipid secretion, which suggests that both bile salts increase the formation of mixed micelles. GDC and TC to a lesser extent increased bile pigment excretion, DHC being without effect. These results favour the hypothesis that micellar binding could be an important factor responsible for the effect of bile acids on bile pigment excretion and should not be completely ruled out.     

Effects of divalent cations and sodium taurocholate on pancreatic lipase activity with gum arabic-emulsified tributyrylglycerol substrates.

The effects of Ca2+ and/or sodium taurocholate on lipase activity with gum arabic-emulsified tributyrylglycerol substrates were investigated. Calcium was found to slightly increase lipase activity while bile salts showed marked inhibition except at very low concentrations. Calcium eliminated inhibition seen with low concentrations of bile salts and reduced the inhibition seen at higher bile shift of the enzyme from the alkaline region in the absence of bile salt to the slightly acidic region in the presence of bile salt. Calcium was shown to eliminate the time lag periods between enzyme addition and maximum rate of hydrolysis seen at low substrate concentrations and the time lag noted when bile salts were included with normal (substrate concentration not limiting) assay concentrations of substrate. Zeta potential measurements indicated that Ca2+ reduced the negative charge on the gum arabic-emulsified particle while bile salts did not increase the negative charge. Commercial preparations of gum arabic were found to have significant concentrations of Ca2+ and Mg2+.     

Effect of free and conjugated bile salts on alpha-amylase activity.

The effect of individual bile salts on alpha-amylase hydrolysis of Cibachron Blue starch was studied at pH 6.0. With sodium cholate, taurocholate and taurodeoxycholate, enzyme activity was increased to 150-160 percent of the control value, at a concentration of similar to 1 mmol/l bile salt. The increased activity extended up to 4 mmol/l. The bile salts sodium deoxycholate and taurochenodeoxycholate exerted activation and inhibition depending on the concentration. With deoxycholate (0.75 mmol/l), activation (150 percent) was evident, while inhibition was apparent above 2.5 mmol/l. With taurochenodeoxycholate maximum activity (135 percent) was observed at 0.25 mmol/l, while inhibition was evident above 1.5 mmol/l. Chenodeoxycholate and lithocholate exerted marked inhibition at concentrations as low as 0.5 mmol/l. Inhibition of alpha-amylase by chenodeoxycholate was competitive with both soluble and insoluble starch substrates. Since the pH of the jejunum is in the region of 6.0 the phenomenon of activation and inhibition of alpha-amylase by bile salts at this pH could be of physiological significance.     

Inhibition of lipases by epsilon-polylysine

Oral administration of epsilon-polylysine to rats reduced the peak plasma triacylglycerol concentration. In vitro, epsilon-polylysine and polylysine strongly inhibited the hydrolysis, by either pancreatic lipase or carboxylester lipase, of trioleoylglycerol (TO) emulsified with phosphatidylcholine (PC) and taurocholate. The epsilon-polylysine concentration required for complete inhibition of pancreatic lipase, 10 microg/ml, is 1,000 times lower than that of BSA required for the same effect. Inhibition requires the presence of bile salt and, unlike inhibition of lipase by other proteins, is not reversed by supramicellar concentrations of bile salt. Inhibition increases with the degree of polylysine polymerization, is independent of lipase concentration, is independent of pH between 5.0 and 9.5, and is accompanied by an inhibition of lipase binding to TO-PC emulsion particles. However, epsilon-polylysine did not inhibit the hydrolysis by pancreatic lipase of TO emulsions prepared using anionic surfactants, TO hydrolysis catalyzed by lingual lipase, or the hydrolysis of a water-soluble substrate. In the presence of taurocholate, epsilon-polylysine becomes surface active and adsorbs to TO-PC monomolecular films. These results are consistent with epsilon-polylysine and taurocholate forming a surface-active complex that binds to emulsion particles, thereby retarding lipase adsorption and triacylglycerol hydrolysis both in vivo and in vitro.     

Nonspecific high affinity binding of bile salts to carboxylester lipases

The interactions with bile salts of carboxylester lipases (EC 3.1.1.13) from human pancreatic juice and pig pancreas were characterized by physical methods. Bile salts cause a decrease in the fluorescence intensity of the proteins at the emission maximum of 333-335 nm. The concentration dependence of this decrease shows saturation behavior, is relatively nonspecific with respect to bile salt conjugation or the presence of the 7 alpha-hydroxyl group, and is consistent with a 1:1 interaction between enzyme and bile salt. Direct measurement of the binding of [3H]cholate by equilibrium dialysis supports the stoichiometry. Other detergents also bind, causing fluorescence changes, but with much lower affinities. Binding of taurocholate to the monomeric pig enzyme is enhanced by increasing ionic strength, indicating the predominance of hydrophobic interactions. In the range of pH 5.5-6.8, binding is pH-independent with dissociation constants of 3-20 microM. At higher pH, affinity is greatly reduced and the fluorescence spectrum changes, indicating the importance of a protonated group for efficient interaction. Occupancy of the bile salt binding site partially stabilizes the enzyme against inactivation by heat but not trypsin. However, circular dichroism spectra do not indicate that bile salt binding is accompanied by any change in secondary structure. The monomeric pig enzyme binds to the argon/water interface in the presence of bile salts and binding of taurocholate to diisopropylphosphoryl-enzyme is similar to that measured with native enzyme. These results suggest that surface binding and catalysis occur at sites distinct from the bile salt binding site of the enzyme. Stabilization of the monomeric pig enzyme against denaturation at high energy surfaces occurs concomitantly with occupancy of the bile salt binding site. Overall, the data suggest that an important role of bile salts in vivo is to stabilize these enzymes at lipid-water interfaces.     

Effects of bile salts and prostaglandins on sodium transport in isolated rat gastric mucosa

To determine whether prostaglandins may protect against bile salt inhibition of ion transport in the stomach, gastric mucosal tissue was isolated from the rat and mounted in flux chambers. Transport of Na+ was traced with radioisotopes in the absence of bile salts and then in the presence of conjugated taurocholate or unconjugated deoxycholate at low, intermediate and high mucosal concentrations (1, 5 and 15 mmol/1). At a high (7.40) or low (3.4) mucosal pH, only the unconjugated deoxycholate inhibited active Na+ transport from mucosa to submucosa with respect to untreated controls. Inhibition of Na+ transport was apparent at a low level of deoxycholate, which also inhibited the electrical potential difference. Intermediate and high levels of deoxycholate lowered the tissue resistance. When the tissues were exposed to mucosal prostaglandin E2 or its 16,16-dimethyl analogue before and during acidified taurocholate administration, Na+ transport was not changed significantly but the electrical resistance remained high. Thus, unconjugated bile salt is more potent than conjugated bile salt in inhibiting Na+ transport and breaking the gastric mucosal barrier, and prostaglandins may afford some small protection.     

Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat

Synthesis of bile salts is regulated through negative feedback inhibition by bile salts returning to the liver. Individual bile salts have not been distinguished with regard to inhibitory potential. We assessed inhibition of bile salt synthesis by either cholate or its taurine conjugate in bile fistula rats. After allowing synthesis to maximize, baseline synthesis was determined by measuring bile salt output in four consecutive 6-hr periods. Next, sodium cholate (+[(14)C]cholate) or taurocholate (+[(14)C]taurocholate) was infused into the jugular vein for 36 hr and bile was collected in 6-hr aliquots. Hepatic flux of exogenous bile salt was determined by measuring output of radioactivity in bile divided by specific activity of the infusate. Synthesis was determined during the last four 6-hr periods of infusion by subtracting exogenous bile salt secretion from the total bile salt output. Thirteen studies using cholate and 13 using taurocholate were performed. Hepatic flux of infused bile salt varied from 1 to 12 micro mol/100 g per rat per hr. Percent suppression of synthesis varied directly with hepatic flux of exogenous bile salt for both cholate and taurocholate in a linear fashion (r = 0.66, P < 0.01 and r = 0.87, P < 0.0005, respectively). Slope of the taurocholate line was 7.82 (% suppression/ micro mol per 100 g per hr), while slope of the cholate line was 3.66 (P < 0.05), indicating that taurocholate was approximately twice as potent as cholate in suppression of synthesis. At fluxes of 10-12 micro mol/100 g per hr, taurocholate suppressed synthesis 84 +/- 8 (SEM) % while cholate suppressed synthesis only 42 +/- 12% (P < 0.02). The x-intercept of the taurocholate line was 0.65 ( micro mol/100 g per hr), while that of the cholate line was -1.01 (NS) suggesting that the threshold for initial suppression of synthesis did not differ for these two bile salts. We conclude that taurocholate is a more effective inhibitor of hepatic bile salt synthesis than cholate, and that intestinal deconjugation of bile salts may play a role in the regulation of synthesis.-Pries, J. M., A. Gustafson, D. Wiegand, and W. C. Duane. Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat.     

Sensitivity of Haloquadratum and Salinibacter to antibiotics and other inhibitors: implications for the assessment of the contribution of Archaea and Bacteria to heterotrophic activities in hypersaline environments

Antibiotics and bile salts have been used to differentiate between heterotrophic activity of halophilic Archaea and Bacteria in saltern ponds. In NaCl-saturated brines of crystallizer ponds, most activity was attributed to Archaea. Following the recent isolation of Haloquadratum, the dominant archaeon in the salterns (reported to be sensitive to chloramphenicol and erythromycin), and the discovery of Salinibacter, a representative of the Bacteria, in the same ecosystem, reevaluation of the earlier data is required. The authors measured amino acid incorporation by Haloquadratum and Salinibacter suspended in crystallizer brine to investigate the suitability of antibiotics and bile salts to distinguish between archaeal and bacterial activities. The amino acid uptake rate per cell in Salinibacter was two orders of magnitude lower than that of Haloquadratum under the same conditions. Salinibacter was inhibited by chloramphenicol, erythromycin, and deoxycholate, but not by taurocholate. Erythromycin did not inhibit incorporation by Haloquadratum, but moderate inhibition was found by chloramphenicol at 10-50 microg mL(-1). Deoxycholate was highly inhibitory, but only partial inhibition was obtained in the presence of 25 microg mL(-1) taurocholate. Inhibition by chloramphenicol and taurocholate increased with increasing salt concentration. Erythromycin and taurocholate proved most valuable to differentiate between archaeal and bacterial activities in saltern brines.     

Acid dissociation constants of bilirubin and related carboxylic acid compounds in bile salt solutions

Bilirubin, the yellow-orange tetrapyrrole pigment of jaundice, is essentially insoluble in pure water, but is much more soluble in solutions of bile salts such as sodium taurocholate. The biophysical chemistry of bilirubin in bile salt solutions is affected by changes in the pH of the solution in the range 5-9, suggesting that interactions with bile salt molecules and micelles may alter the acidity of the pigment. We have examined this possibility by determining the apparent pKa values for a series of carboxyl 13C-enriched model compounds, including the bilirubin analog mesobilirubin XIIIalpha, in solutions of sodium taurocholate and sodium taurodeoxycholate. Apparent pKa values were determined by 13C NMR titrations in dimethyl sulfoxide-water mixtures. The results show that the acidity of all compounds is decreased, or pKa increased, in micellar bile salt solution relative to pure water and that the effect is greatest for the larger, less water-soluble compounds. We have proposed a model to explain these results and discussed the implications of these findings for the biophysical chemistry of bilirubin in bile.     

The effect of bile salts on oxygen consumption, oxidative phosphorylation and ATP-ase activity of mucosal homogenates from rat jejunum and ileum

The conjugated bile salts, sodium taurocholate and glycocholate, inhibited oxygen consumption and uncoupled oxidative phosphorylation of mucosal homogenates from rat jejunum and ileum. These bile salts also were capable of increasing the ATP-ase activity, in the presence of Na⁺ + K⁺ with Mg⁺⁺, of both mucosal homogenates. Consequently, it was concluded from the results of this investigation that the previously observed decrease in ATP levels of rat jejunum and ileum, in the presence of bile salts, can be accounted for by both a complete uncoupling of oxidative phosphorylation and by an increase in ATP-ase activity. Furthermore, the mechanism of bile salt inhibition of tissue ATP levels was discussed in relation to a regulatory role played by bile salts in the active transport of water soluble substances across the small intestine.     

Interactions of cationic bile salt derivatives with the ileal bile salt transport system.

Previous structure-activity studies of the active ileal bile salt transport system have demonstrated that a single negative charge on the side chain is essential for active transport. Furthermore, mutual inhibition studies between different pairs of bile salt substrates indicated that dihydroxy bile salts had a greater apparent affinity for the transport system than the trihydroxylated compounds and triketo bile salts had the least such affinity. In this study, a series of cationic bile salt derivatives (cholamine conjugates) were prepared with one, two, and three alpha-hydroxyl groups on the steroid moiety. Based on the previous observations one would expect (1) no active transport of any of the cholamine conjugates by the ileal transport system; (2) interaction of these compounds with the transport system in such a way as to inhibit the transport of bile salts, with inhibition potency of the transport of any single bile salt inversely related to the number of hydroxyl groups present on the cholamine conjugate; and (3) transport of triketo anionic bile salts to be most readily inhibited, trihydroxy compounds less readily inhibited, and dihydroxy bile salts least inhibited. Using everted gut sac preparations it was demonstrated that all three aforementioned expectations did occur. Furthermore, reversible inhibition of ileal absorption of taurocholate and the bile salt derivative taurodehydrocholate could be demonstrated in vivo. The dihydroxy cholamine conjugates were better inhibitors than the trihydroxy compound. Relative specificity for the bile salt system of these cationic bile salt derivatives was demonstrated in the in vivo preparation by comparing its inhibition of taurodehydrocholate absorption with their lesser capacity to inhibit glucose transport.     

Bile salt excretion in skate liver is mediated by a functional analog of Bsep/Spgp, the bile salt export pump

Biliary secretion of bile salts in mammals is mediated in part by the liver-specific ATP-dependent canalicular membrane protein Bsep/Spgp, a member of the ATP-binding cassette superfamily. We examined whether a similar transport activity exists in the liver of the evolutionarily primitive marine fish Raja erinacea, the little skate, which synthesizes mainly sulfated bile alcohols rather than bile salts. Western blot analysis of skate liver plasma membranes using antiserum raised against rat liver Bsep/Spgp demonstrated a dominant protein band with an apparent molecular mass of 210 kDa, a size larger than that in rat liver canalicular membranes, approximately 160 kDa. Immunofluorescent localization with anti-Bsep/Spgp in isolated, polarized skate hepatocyte clusters revealed positive staining of the bile canaliculi, consistent with its selective apical localization in mammalian liver. Functional characterization of putative ATP-dependent canalicular bile salt transport activity was assessed in skate liver plasma membrane vesicles, with [(3)H]taurocholate as the substrate. [(3)H]taurocholate uptake into the vesicles was mediated by ATP-dependent and -independent mechanisms. The ATP-dependent component was saturable, with a Michaelis-Menten constant (K(m)) for taurocholate of 40+/-7 microM and a K(m) for ATP of 0.6+/-0.1 mM, and was competitively inhibited by scymnol sulfate (inhibition constant of 23 microM), the major bile salt in skate bile. ATP-dependent uptake of taurocholate into vesicles was inhibited by known substrates and inhibitors of Bsep/Spgp, including other bile salts and bile salt derivatives, but not by inhibitors of the multidrug resistance protein-1 or the canalicular multidrug resistance-associated protein, indicating a distinct transport mechanism. These findings provide functional and structural evidence for a Bsep/Spgp-like protein in the canalicular membrane of the skate liver. This transporter is expressed early in vertebrate evolution and transports both bile salts and bile alcohols.     

Role of the hepatocyte microtubular system in the excretion of bile salts and biliary lipid: implications for intracellular vesicular transport

The role of the hepatocyte microtubular system in the transport and excretion of bile salts and biliary lipid has not been defined. In this study the effects of microtubule inhibition on biliary excretion of micelle- and non-micelle-forming bile salts and associated lipid were examined in rats. Low-dose colchicine pretreatment had no effect on the baseline excretion of biliary bile salts and phospholipid in animals studied 1 hr after surgery (basal animals), but slightly retarded the excretion of tracer [14C]taurocholate relative to that of lumicolchicine-pretreated (control) rats. However, colchicine pretreatment resulted in a marked reduction in the excretion of 2 mumol/100 g doses of a series of four micelle-forming bile salts of differing hydrophilicity, but had no significant effect on the excretion of the non-micelle-forming bile salt, taurodehydrocholate. Continuous infusion of 0.2 mumol of taurocholate/(100 g.min) following 24 hr of biliary drainage (depleted/reinfused animals) resulted in physiologic bile flow with biliary excretion rates of bile salts, phospholipid, and cholesterol that were markedly inhibited (mean 33, 39, and 42%, respectively) by colchicine or vinblastine pretreatment. Excretion of tracer [14C]taurocholate also was markedly delayed by colchicine in these bile salt-depleted/reinfused animals. In contrast, colchicine did not inhibit bile salt excretion in response to reinfusion of taurodehydrocholate. Thus, under basal conditions, the microtubular system appears to play a minor role in hepatic transport and excretion of bile salts and biliary lipid. However, biliary excretion of micelle-forming bile salts and associated phospholipid and cholesterol becomes increasingly dependent on microtubular integrity as the transcellular flux and biliary excretion of bile salts increases, in both bile salt-depleted and basal animals. We postulate that cotransport of micelle-forming bile salts and lipids destined for biliary excretion, via an intracellular vesicular pathway, forms the basis for this microtubule dependence.     

Estimation of the contribution of halobacterial to the bacterial biomass and activity in solar salterns by the use of bile salts

Abstract Bile salts (deoxycholate, taurocholate) were used to estimate the contribution of bacteria of the Halobacterium group to bacterial community size and activity at different salinities as found in a multi-pond saltern. Low concentrations of bile salts cause lysis of halophilic archaebacteria of the Halobacterium group, while halophilic eubacteria and halococci remain microscopically intact. Upon addition of bile salts, total bacterial numbers (as estimated microscopically) in saltern ponds at salinities below 250 g/l did not decrease, while above this salinity bacterial numbers decreased by 30–50%. To estimate the contribution of halobacteria to overall heterotrophic activity, the effect of bile salt addition was tested on the incorporation of labelled amino acids. In saltern ponds of a salinity below 250 g/l activity was not greatly inhibited by taurocholate, while at salinity above 300 g/l taurocholate completely abolished incorporation of amino acids.     

Investigations on the sodium dependence of bile acid fluxes in the isolated perfused rat liver.

At [Na+]o = 118 mM the concentrative transfer of cholic and taurocholic acid from the perfusate into the isolated rat liver displays saturation kinetics (taurocholate: V = 299 nmol-min-1-g-1, Km = 61 muM; Cholate: V=327 nmol-min-1-g-1, Km = 436 muM). Perfusion with an isotonic sodium-free medium did not change the feature of a carrier-mediated transport but did markedly reduce V without affecting Km (taurocholate: V = 65 nmol-min-1-g-1, Km = 78 muM; cholate: V = 104 nmol-min-1-g-1, Km = 354 muM). It was experimentally assured that the observed reduction of bile salt uptake was not a consequence of regurgitation of bile salts or due to an excessive intracellular accumulation during cholestasis in the sodium-free state. The rate of taurocholate efflux is very low when compared with the rapid rate of the uptake. A stimulatory action of extracellular sodium on this pathway was also observed. Inhibition of the (Na+ + K+)-ATPase by 1 mM ouabain resulted in a decrease of bile salt uptake. Activation of the enzyme by potassium readmission to a K+-deprived liver enhanced bile salt uptake. The immediate response to alteration of the enzyme activity suggests a close association of a fraction of bile acid active transport with the sodium pump.     

An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts

F(+) strains of Escherichia coli infected with donor-specific bacteriophage such as M13 are sensitive to bile salts. We show here that this sensitivity has two components. The first derives from secretion of bacteriophage particles through the cell envelope, but the second can be attributed to expression of the F genes required for the formation of conjugative (F) pili. The latter component was manifested as reduced or no growth of an F(+) strain in liquid medium containing bile salts at concentrations that had little or no effect on the isogenic F(-) strain or as a reduced plating efficiency of the F(+) strain on solid media; at 2% bile salts, plating efficiency was reduced 10(4)-fold. Strains with F or F-like R factors were consistently more sensitive to bile salts than isogenic, plasmid-free strains, but the quantitative effect of bile salts depended on both the plasmid and the strain. Sensitivity also depended on the bile salt, with conjugated bile salts (glycocholate and taurocholate) being less active than unconjugated bile salts (deoxycholate and cholate). F(+) cells were also more sensitive to sodium dodecyl sulfate than otherwise isogenic F(-) cells, suggesting a selectivity for amphipathic anions. A mutation in any but one F tra gene required for the assembly of F pili, including the traA gene encoding F pilin, substantially restored bile salt resistance, suggesting that bile salt sensitivity requires an active system for F pilin secretion. The exception was traW. A traW mutant was 100-fold more sensitive to cholate than the tra(+) strain but only marginally more sensitive to taurocholate or glycocholate. Bile salt sensitivity could not be attributed to a generalized change in the surface permeability of F(+) cells, as judged by the effects of hydrophilic and hydrophobic antibiotics and by leakage of periplasmic beta-lactamase into the medium.     

Effects of bile salts on the plasma membranes of isolated rat hepatocytes.

The conjugated trihydroxy bile salts glycocholate and taurocholate removed approx. 20--30% of the plasma-membrane enzymes 5'-nucleotidase, alkaline phosphatase and alkaline phosphodiesterase I from isolated hepatocytes before the onset of lysis, as judged by release of the cytosolic enzyme lactate dehydrogenase. The conjugated dihydroxy bile salt glycodeoxycholate similarly removed 10--20% of the 5'-nucleotidase and alkaline phosphatase activities, but not alkaline phosphodiesterase activity; this bile salt caused lysis of hepatocytes at approx. 10-fold lower concentrations (1.5--2.0mM) than either glycocholate or taurocholate (12--16mM). At low concentrations (7 mM), glycocholate released these enzymes in a predominantly particulate form, whereas at higher concentrations (15 mM) glycocholate further released these components in a predominantly 'soluble' form. Inclusion of 1% (w/v) bovine serum albumin in the incubations had a small protective effect on the release of enzymes from hepatocytes by glycodeoxycholate, but not by glycocholate. These observations are discussed in relation to the possible role of bile salts in the origin of some biliary proteins.     

Inhibition of lipase adsorption at interfaces. Role of bile salt micelles and colipase

The effects of bile salts and colipase on the adsorption of lipase at an interface were studied by hydrophobic affinity chromatography on phenyl- and octyl-Sepharose. In the absence of bile salts, lipase or colipase binds separately to the gel. This is unchanged in the presence of adsorbed bile salts, when one bile salt molecule is associated per hydrophobic ligand. The same data are obtained in the presence of monomeric bile salt solutions. In contrast, lipase adsorption is totally prevented in a micellar bile salt solution. These results favor the idea that the formation of a lipase-bile salt complex in solution is responsible for the lack of interfacial lipase adsorption.     

Calcium binding by monosulfate esters of taurochenodeoxycholate

The effect of sulfate esterification of the 3 alpha- or 7 alpha-hydroxyl groups of taurochenodeoxycholate on calcium binding was studied at 0.154 M NaCl in the presence and absence of phosphatidylcholine using a calcium electrode. For comparison, similar studies were made with taurochenodeoxycholate, taurodeoxycholate, and taurocholate. No high affinity calcium binding was demonstrable for any of these bile salts in pre-micellar solutions. Taurine-conjugated bile salts have greater affinity for calcium when in a micellar form. At elevated bile salt concentrations, the calcium binding of unsulfated dihydroxy taurine conjugates was similar to that of the monosulfate esters of taurochenodeoxycholate. The presence of phosphatidylcholine decreased calcium binding of the unsulfated dihydroxy bile salts and slightly increased calcium binding by taurocholate. However, the addition of phosphatidylcholine to monosulfate esters of taurochenodeoxycholate results in large increments in calcium binding. The results indicate that increased calcium binding due to the presence of phosphatidylcholine in bile salt solutions depends, in part, on the hydrophilicity of the bile salt and that the interaction of monosulfate esters of taurochenodeoxycholate with phosphatidylcholine leads to the formation of a high affinity calcium binding site.